The threat of brucellosis is of particular concern because of its potential to disrupt agricultural economy, and the disease continues to be problematic in parts of the U.S. today. To compound the problem, elk and wild bison within the Greater Yellowstone Area (GYA) are major reservoirs for brucellosis and cattle may contract Brucella abortus from this population (Godfroid, J. (2002) Rev. Sci. Technol. Off. Int. Epiz., 21:277-286). Wildlife Brucella reservoirs represent a major obstacle to the development of an effective eradication program focusing on domestic livestock in Wyoming. Therefore, the Wyoming Brucellosis Coordination Team has issued a series of management practices aimed at controlling brucellosis in elk. Among these practices is a recommendation to test feed ground elk as a way to monitor sero-prevalence and efficacy of brucellosis elimination activities.
Unfortunately, diagnostic methods for brucellosis have been limited because of the lack of consistently reliable targets which ensure high specificity and sensitivity. More sensitive than traditional Brucella diagnostic methods, serologic diagnosis based on reactivity to LPS has been reported (Saegerman et al. (2004) Vet. Microbiol., 100:91-105). Geographic areas of false positive serologic reactions exist however, which reduce specificity of such assays (Saegerman et al. (2004) Vet. Microbiol., 100:91-105). More recently, PCR-based tests have been evaluated as a next-generation approach to early diagnosis/detection, although standardization of methodologies and a more diverse repertoire of target genes still need to be established (Al Dahouk et al. (2004) Clin. Lab., 50:387-394; Navarro et al. (2004) Exp. Rev. Mol. Diag., 4:115-123).
While several genes and their products associated with Brucella virulence have been described (for review, see Ko et al. (2003) Clin. Microbiol. Rev., 16:65-78), most have been identified using in vitro-grown bacteria. In this approach, host factors important in up-regulating some virulence loci may not be present in laboratory-grown cultures. Signature Tagged Mutagenesis (STM) has been employed with Brucella spp. in an attempt to identify virulence genes which are requisite to survival in vivo (Hong et al. (2000) Infect. Immun., 68:4102-4107; Zygmunt et al. (2006) Microb. Infect., 8:2849-2854). This technique involves a “negative” selection approach which relies on live animals to only identify mutations in those genes essential for host survival, and not immunogenic gene products. The method is also quite sensitive to experimental variables and to date has yielded limited information on molecular aspects of Brucella virulence. A less cumbersome and less artifactual approach is to utilize immune sera adsorbed with the in vitro-grown pathogen as a screening reagent for those gene products relevant to in vivo survival and pathogenesis. This technique is known as In vivo-Induced Antigen Technology, (IVIAT), and has been successfully used on bacterial pathogens to identify antigenic proteins expressed during infection (Handfield et al. (2000) Trends Microbiol., 8:336-339; Rollins et al. (2005) Cell. Microbiol., 7:1-9). Most recently, IVIAT has been applied to Bacillus anthracis to identify potential diagnostic, vaccine, and therapeutic candidates (Rollins et al. (2008) PLoS One 3:e1824). IVIAT has also been used with other facultative intracellular pathogens, such as Mycobacterium tuberculosis (Deb et al. (2002) Tuberculosis 82:175-182) and Legionella pneumophila (Chang et al. (2005) Infect. Immun., 73:4272-4280).